Field of the Invention
This invention relates to surface plasmon field-enhanced fluorescence spectroscopy (SPFS), and in particular, it relates to SPFS biosensors based on nucleic acid ligand structural change.
Description of Related Art
Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) is a known biosensing technology. The technology is described in, for example, T. Liebermann, W. Knoll, Surface-plasmon field-enhanced fluorescence spectroscopy, Colloids and Surfaces A: Physicochem. Eng. Aspects 171 (2000) 115-130 (“Liebermann 2000”); and Wolfgang Knoir, Fang Yu, Thomas Neumann, Lifang Niu, and Evelyne L. Schmid, Principles And Applications Of Surface Plasmon Field-Enhanced Fluorescence Techniques, in Topics in Fluorescence Spectroscopy, Volume 8: Radiative Decay Engineering, Edited by Geddes and Lakowicz, Springer Science+Business Media, Inc., New York, 2005, p. 305-332. These references show the principle and setup of SPFS biosensors in general. SPFS offers high-sensitivity detection through advanced sensing technology.
FIG. 1A, taken from FIG. 5 of the Liebermann 2000 paper, illustrates the setup of an SPFS system. FIG. 1B, taken from FIG. 6(a) of the same paper, illustrates the structure of the prism and flow cell used in the SPFS system. The basic concept of SPFS is described below with reference to FIGS. 1, 1A and 1B. An SPFS biosensor includes a thin metal film on a glass or plastic prism. The metal may be, for example, gold, silver, aluminum, etc. A capture molecule is immobilized on the surface of the metal film. A biological sample is applied on the metal film. When an incident light of a certain wavelength is irradiated on the prism at a certain angle, a relatively strong electrical field is generated at the surface of the metal film. Because of quenching from the metal film, the best place for fluorescence excitation is in the region about a couple of tens to hundreds nm above the surface. In a typical device, the quenching region is within about 0-5 nm from the metal surface, and the enhanced region is about 10-200 nm from the surface. If a fluorescent label is trapped in this enhanced region, a relatively strong fluorescent signal is generated.
SPFS biosensors are based on fluorescence detection. In conventional SPFS biosensors, in addition to first antibodies that are immobilized on the thin metal film, fluorescent labeled second antibodies are generally used for protein detection. This is schematically illustrated in FIG. 1. The first antibodies 15 are immobilized on the thin metal film 11 on the prism 12. The target 17 (i.e. substance to be detected, such as protein) are added to the biosensor and captured on the immobilized first antibodies 15. Then, the fluorescent labeled second antibodies 16 are added to the biosensor and they bind to the target 17. The first antibody 15, the target 17 and the second antibody 16 form a structure such that the fluorescent label 16F on the second antibody is located in the region of enhanced electric field above the thin metal film 11, and a relatively strong fluorescent signal is generated. For unbound second antibodies or those that form non-specific binding, their fluorescent labels tend to be located outside of the enhanced region, in the metal quenching region or farther away from the surface, so they are not excited. The biosensor can be washed before the detection result is obtained. These multiple steps make the biosensor more complicated to use and the turnaround time long.